Ixodes scapularis ticks transmit Borrelia burgdorferi (agent of Lyme disease), Anaplasma phagocytophilum (agent of human granulocytic ehrlichiosis) and Babesia microti (agent of human babesiosis) among other pathogens. A. phagocytophilum and B. microti infect the salivary glands of the tick, hence poised to exit the tick vector during feeding. On the other hand, B. burgdorferi colonizes the tick midgut, anchoring to the midgut via a protein-protein interaction involving the tick midgut protein TROSPA and the spirochete lipoprotein OspA. The commencement of feeding, provides cues to the spirochete that trigger active growth and replication of the bacterium, as it prepares to migrate from the midgut through the haemolymph to the salivary glands, the port of exit to the vertebrate host. While there is a growing understanding of the tick salivary gland transcriptome and its dynamic influence on pathogen transmission, little is known about the midgut proteome and how Borrelia interacts with the midgut proteins during the critical phase of growth and migration. Borrelia is known to interact with tick and host proteins during its life cycle to disseminate and to survive in hostile and diverse milieus. While critical non-protein interactions are also expected to play a role in transmission, this proposal will focus on protein interactions between the spirochete and tick midgut. We postulate that B. burgdorferi may interact with I. scapularis midgut proteins to facilitate its growth and migration from the midgut;and that these tick proteins may offer novel targets to block the transmission of the Lyme disease pathogen. With the goal of testing this postulate, we will utilize a yeast display approach to define tick midgut proteins that specifically interact with the spirochete. RNA interference technique will be exploited to determine if the interaction facilitates the events that precede transmission to the vertebrate host. These events include Borrelia replication, survival, egress from the midguts, migration through the haemocoel and entry into salivary glands. Research efforts by various groups have demonstrated that the proteome of B. burgdorferi undergoes dramatic changes during the process of spirochete growth in the tick midgut, changes influenced perhaps by changes in temperature, pH or other as yet undefined factors in the tick midgut. In this proposal we will define if interactions between the spirochete and specific midgut proteins signals changes in spirochete gene expression. These studies will provide new insights into the dynamics of the interaction between the tick and the spirochete and how these interactions influence spirochete transmission. These studies, when completed will also provide a powerful tool to address vector-pathogen and host-pathogen interactions in other disease models of high public health importance. Importantly, these studies will reveal a hitherto unexplored facet of gene regulation in Borrelia burgdorferi and promote a greater understanding of Lyme disease pathogenesis. PUBLIC HEALTH RELEVANCE Ixodes scapularis ticks transmit Borrelia burgdorferi, the agent of Lyme disease among other pathogens. In this proposal we will identify tick midgut proteins that interact with Borrelia during spirochete growth and migration and examine the impact of this interaction on Borrelia growth in the tick and transmission to the murine host.